Target: IEC224 Phosphoserine phosphatase NCBI Gene #: 11915261 Protein ID#: YP_005334106.1 (Used Dax's Oligo Design to order primers. Couldn't find Will's DNA Works Output on GDocs. Julia's first primer is small..? - Dr. B 090313)
organism: Vibrio cholerae etiologic risk group: Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia
Background/Disease Information (sort of like the Intro to your Mini Research Write up): Vibrio cholerae is a bacteria that can cause a disease known as cholera depending on the strain encountered. The disease was first discovered in 1817 when it was spread by using trading routes. Several pandemics have occured through the mass spreading of the bacteria. In addition to several antibacterial treatments for extreme cases of the disease, vaccines are available to prevent transmission. However, the main treatment used is a process called oral rehydration therapy.
Cholera is spread when a host consumes food or drink that has been contaminated with the waste of an infected person. Some of the main symptoms include diarrhea and vomiting. Symptoms typically occur between one and five days from becoming infected. Water is usually the cause of cholera in developing countries and although rare, the cause of cholera in developed regions of the world is seafood particularly plankton and shellfish. The nickname for this disease is "blue death" due to it causing blue rashes on the body of an infected person. Currently, there are around 3-5 million people experiencing cholera worldwide.
To become infected, the average ingestion of the bacteria must be at around 100 million. Of course this number is lowered in people with weak immune systems and certain diseases. Some of the most infected parts of the world include India, Haiti, certain parts of Africa, and a few others.
.
Essentiality of this protein: essential Complex of proteins?: works alone with current information available Druggable Target: yes
Current Inhibitors: none found Expression Information (has it been expressed in bacterial cells): under Brenda, this enzyme has been expressed in porphyromonas gingivalis Purification Method: http://www.sciencedirect.com.ezproxy.lib.utexas.edu/science/article/pii/S0014299997013046?np=y Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSLDALTTLPIKKHTALLNRFPETRFVTQLAKKRASWIVFGHYLTPAQFEDMDFFTNRFNAILDMWKVGRYEVALMDGEL TSEHETILKALELDYARIQDVPDLTKPGLIVLDMDSTAIQIECIDEIAKLAGVGEEVAEVTERAMQGELDFEQSLRLRVS KLKDAPEQILSQVRETLPLMPELPELVATLHAFGWKVAIASGGFTYFSDYLKEQLSLDYAQSNTLEIVSGKLTGQVLGEV VSAQTKADILLTLAQQYDVEIHNTVAVGDGANDLVMMAAAGLGVAYHAKPKVEAKAQTAVRFAGLGGVVCILSAALVAQQ KLSWKSKEGHHHHHH
Julia C. Dr. B said that we should use the NCBI sequence instead of the PDB one. Here's the 'correct' filtered through aa sequence: mdalttlpikkhtallnrfpetrfvtqlakkraswivfghyltpaqfedmdfftnrfnai ldmwkvgryevalmdgeltsehetilkaleldyariqdvpdltkpglivldmdstaiqie cideiaklagvgeevaevteramqgeldfeqslrlrvsklkdapeqilsqvretlplmpe lpelvatlhafgwkvaiasggftyfsdylkeqlsldyaqsntleivsgkltgqvlgevvs aqtkadilltlaqqydveihntvavgdgandlvmmaaaglgvayhakpkveakaqtavrf aglggvvcilsaalvaqqklswkskp
*length of your protein in Amino Acids: 335 (Julia C.: 326) Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 36981.5 Da Molar Extinction coefficient of your protein at 280 nm wavelength: 34045 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only):
atgagcctggatgcgctgaccaccctgccgattaaaaaacataccgcgctgctgaaccgc tttccggaaacccgctttgtgacccagctggcgaaaaaacgcgcgagctggattgtgttt ggccattatctgaccccggcgcagtttgaagatatggatttttttaccaaccgctttaac gcgattctggatatgtggaaagtgggccgctatgaagtggcgctgatggatggcgaactg accagcgaacatgaaaccattctgaaagcgctggaactggattatgcgcgcattcaggat gtgccggatctgaccaaaccgggcctgattgtgctggatatggatagcaccgcgattcag attgaatgcattgatgaaattgcgaaactggcgggcgtgggcgaagaagtggcggaagtg accgaacgcgcgatgcagggcgaactggattttgaacagagcctgcgcctgcgcgtgagc aaactgaaagatgcgccggaacagattctgagccaggtgcgcgaaaccctgccgctgatg ccggaactgccggaactggtggcgaccctgcatgcgtttggctggaaagtggcgattgcg agcggcggctttacctattttagcgattatctgaaagaacagctgagcctggattatgcg cagagcaacaccctggaaattgtgagcggcaaactgaccggccaggtgctgggcgaagtg gtgagcgcgcagaccaaagcggatattctgctgaccctggcgcagcagtatgatgtggaa attcataacaccgtggcggtgggcgatggcgcgaacgatctggtgatgatggcggcggcg ggcctgggcgtggcgtatcatgcgaaaccgaaagtggaagcgaaagcgcagaccgcggtg cgctttgcgggcctgggcggcgtggtgtgcattctgagcgcggcgctggtggcgcagcag aaactgagctggaaaagcaaagaaggccatcatcatcatcatcat
Dax: This is the gene sequence I found, and what was used for primer tail design and ordering. It has a 100% query coverage and 88% match to Julia's sequence, but when put in an amino acid sequence, the two match at 100% for both query coverage and identity.
*GC% Content for gene: 56.3% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): Upstream: TACTTCCAATCCATGGACGCGCTGACCA Downstream (reverse complemented): TATCCACCTTTACTGTTACGGTTTAGATTTCCAAGAGAG *Note that this insert contains code that codes for a cut by BsaI.
NCBI Gene #: 11915261
Protein ID#: YP_005334106.1 (Used Dax's Oligo Design to order primers. Couldn't find Will's DNA Works Output on GDocs. Julia's first primer is small..? - Dr. B 090313)
organism: Vibrio cholerae
etiologic risk group: Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia
Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Vibrio cholerae is a bacteria that can cause a disease known as cholera depending on the strain encountered. The disease was first discovered in 1817 when it was spread by using trading routes. Several pandemics have occured through the mass spreading of the bacteria. In addition to several antibacterial treatments for extreme cases of the disease, vaccines are available to prevent transmission. However, the main treatment used is a process called oral rehydration therapy.
Cholera is spread when a host consumes food or drink that has been contaminated with the waste of an infected person. Some of the main symptoms include diarrhea and vomiting. Symptoms typically occur between one and five days from becoming infected. Water is usually the cause of cholera in developing countries and although rare, the cause of cholera in developed regions of the world is seafood particularly plankton and shellfish. The nickname for this disease is "blue death" due to it causing blue rashes on the body of an infected person. Currently, there are around 3-5 million people experiencing cholera worldwide.
To become infected, the average ingestion of the bacteria must be at around 100 million. Of course this number is lowered in people with weak immune systems and certain diseases. Some of the most infected parts of the world include India, Haiti, certain parts of Africa, and a few others.
.
Essentiality of this protein: essential
Complex of proteins?: works alone with current information available
Druggable Target: yes
*EC#: 3.1.3.3
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.3
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/catalog/search?interface=All&term=phosphoserine&lang=en®ion=US&focus=product&N=0+220003048+219853269+219853286
-- link (or citation) to paper that contains assay information
http://www.sciencedirect.com.ezproxy.lib.utexas.edu/science/article/pii/S0014299997013046?np=y
-- List cost and quantity of substrate reagents and supplier
phosphoserine 63.50
Structure Available (PDB or Homology model)
http://www.rcsb.org/pdb/explore/explore.do?structureId=3N28
Current Inhibitors: none found
Expression Information (has it been expressed in bacterial cells): under Brenda, this enzyme has been expressed in porphyromonas gingivalis
Purification Method:
http://www.sciencedirect.com.ezproxy.lib.utexas.edu/science/article/pii/S0014299997013046?np=y
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSLDALTTLPIKKHTALLNRFPETRFVTQLAKKRASWIVFGHYLTPAQFEDMDFFTNRFNAILDMWKVGRYEVALMDGEL TSEHETILKALELDYARIQDVPDLTKPGLIVLDMDSTAIQIECIDEIAKLAGVGEEVAEVTERAMQGELDFEQSLRLRVS KLKDAPEQILSQVRETLPLMPELPELVATLHAFGWKVAIASGGFTYFSDYLKEQLSLDYAQSNTLEIVSGKLTGQVLGEV VSAQTKADILLTLAQQYDVEIHNTVAVGDGANDLVMMAAAGLGVAYHAKPKVEAKAQTAVRFAGLGGVVCILSAALVAQQ KLSWKSKEGHHHHHH
Julia C.
Dr. B said that we should use the NCBI sequence instead of the PDB one. Here's the 'correct' filtered through aa sequence:
mdalttlpikkhtallnrfpetrfvtqlakkraswivfghyltpaqfedmdfftnrfnai ldmwkvgryevalmdgeltsehetilkaleldyariqdvpdltkpglivldmdstaiqie cideiaklagvgeevaevteramqgeldfeqslrlrvsklkdapeqilsqvretlplmpe lpelvatlhafgwkvaiasggftyfsdylkeqlsldyaqsntleivsgkltgqvlgevvs aqtkadilltlaqqydveihntvavgdgandlvmmaaaglgvayhakpkveakaqtavrf aglggvvcilsaalvaqqklswkskp
*length of your protein in Amino Acids: 335 ( Julia C.: 326)
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 36981.5 Da
Molar Extinction coefficient of your protein at 280 nm wavelength: 34045
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
atgagcctggatgcgctgaccaccctgccgattaaaaaacataccgcgctgctgaaccgc tttccggaaacccgctttgtgacccagctggcgaaaaaacgcgcgagctggattgtgttt ggccattatctgaccccggcgcagtttgaagatatggatttttttaccaaccgctttaac gcgattctggatatgtggaaagtgggccgctatgaagtggcgctgatggatggcgaactg accagcgaacatgaaaccattctgaaagcgctggaactggattatgcgcgcattcaggat gtgccggatctgaccaaaccgggcctgattgtgctggatatggatagcaccgcgattcag attgaatgcattgatgaaattgcgaaactggcgggcgtgggcgaagaagtggcggaagtg accgaacgcgcgatgcagggcgaactggattttgaacagagcctgcgcctgcgcgtgagc aaactgaaagatgcgccggaacagattctgagccaggtgcgcgaaaccctgccgctgatg ccggaactgccggaactggtggcgaccctgcatgcgtttggctggaaagtggcgattgcg agcggcggctttacctattttagcgattatctgaaagaacagctgagcctggattatgcg cagagcaacaccctggaaattgtgagcggcaaactgaccggccaggtgctgggcgaagtg gtgagcgcgcagaccaaagcggatattctgctgaccctggcgcagcagtatgatgtggaa attcataacaccgtggcggtgggcgatggcgcgaacgatctggtgatgatggcggcggcg ggcctgggcgtggcgtatcatgcgaaaccgaaagtggaagcgaaagcgcagaccgcggtg cgctttgcgggcctgggcggcgtggtgtgcattctgagcgcggcgctggtggcgcagcag aaactgagctggaaaagcaaagaaggccatcatcatcatcatcat
Julia C.
The gene sequence I found:
1 ATGGACGCGCTGACCACGCTGCCGATCAAAAAACACACCGCGCTCCTGAACCGTTTCCCG
61 GAAACCCGTTTCGTTACGCAACTGGCGAAGAAACGCGCGAGCTGGATCGTTTTCGGTCAC
121 TATCTGACCCCGGCACAGTTTGAAGATATGGACTTCTTCACCAATCGCTTTAATGCCATC
181 CTCGATATGTGGAAAGTGGGTCGTTATGAGGTTGCGCTCATGGACGGTGAACTCACCTCT
241 GAACACGAAACCATTCTGAAGGCGCTGGAACTCGATTACGCACGTATCCAGGACGTTCCG
301 GACCTCACCAAACCGGGTCTCATCGTTCTGGACATGGATTCTACCGCGATTCAGATCGAA
361 TGCATCGACGAAATCGCGAAGCTGGCGGGTGTCGGTGAGGAAGTTGCGGAAGTTACCGAA
421 CGTGCTATGCAGGGCGAACTGGATTTCGAACAGTCTCTGCGTCTCCGTGTTTCTAAACTG
481 AAGGATGCACCGGAACAGATCCTGAGCCAAGTTCGTGAAACCCTGCCGCTGATGCCGGAA
541 CTGCCAGAGCTCGTTGCGACCCTGCACGCATTCGGTTGGAAGGTAGCAATCGCCTCCGGT
601 GGTTTTACCTACTTCAGCGACTACCTGAAAGAGCAGCTCTCTCTGGACTATGCGCAGTCT
661 AACACCCTCGAAATTGTTTCTGGTAAACTCACTGGTCAGGTTCTCGGTGAAGTTGTCTCC
721 GCGCAGACCAAAGCGGACATCCTGCTGACGCTCGCCCAGCAGTATGACGTTGAAATTCAC
781 AACACCGTTGCGGTTGGCGATGGCGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTG
841 GGTGTGGCCTACCACGCGAAACCGAAAGTCGAAGCAAAGGCGCAAACGGCGGTTCGTTTT
901 GCGGGTCTGGGTGGTGTCGTTTGTATTCTGTCTGCCGCGCTGGTGGCGCAGCAAAAACTG
961 TCTTGGAAATCTAAACCGTAA
Dax:
This is the gene sequence I found, and what was used for primer tail design and ordering. It has a 100% query coverage and 88% match to Julia's sequence, but when put in an amino acid sequence, the two match at 100% for both query coverage and identity.
ATGGACGCGCTGACCACCCTCCCGATCAAAAAGCACACCGCGCTGCTGAACCGTTTC CCGGAAACCCGCTTCGTTACCCAACTGGCGAAAAAGCGTGCGTCTTGGATCGTTTTCGGTCAC
TACCTCACTCCAGCACAGTTTGAAGATATGGATTTTTTCACCAATCGTTTCAATGCGATC
CTGGACATGTGGAAAGTTGGCCGTTACGAAGTTGCGCTGATGGACGGTGAACTGACCTCT
GAACACGAAACCATCCTGAAAGCGCTGGAACTCGACTACGCTCGCATCCAGGACGTTCCA
GACCTCACCAAACCGGGCCTGATCGTTCTCGACATGGACTCTACCGCTATCCAGATCGAA
TGCATCGACGAAATTGCGAAGCTGGCGGGTGTTGGCGAGGAAGTGGCCGAAGTTACGGAA
CGTGCGATGCAGGGCGAGCTGGACTTCGAACAGTCTCTGCGTCTGCGTGTTTCTAAACTC
AAAGACGCCCCTGAACAGATCCTGAGCCAGGTTCGTGAAACGCTGCCGCTCATGCCTGAA
CTGCCGGAACTGGTTGCGACCCTGCACGCGTTCGGTTGGAAGGTAGCAATCGCGTCTGGT
GGTTTCACCTACTTTTCTGACTACCTGAAGGAACAACTCAGCCTCGATTACGCGCAGTCT
AACACCCTGGAAATTGTTTCTGGTAAACTGACTGGTCAAGTTCTGGGTGAAGTTGTGTCT
GCTCAGACCAAAGCGGACATCCTGCTGACCCTGGCGCAACAGTACGACGTTGAAATCCAC
AACACCGTTGCGGTGGGTGACGGTGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTC
GGTGTAGCGTACCATGCGAAACCGAAGGTTGAGGCGAAGGCGCAGACCGCAGTTCGTTTC
GCTGGTCTCGGTGGTGTCGTTTGCATCCTGTCTGCGGCGCTCGTTGCGCAGCAAAAACTC
TCTTGGAAATCTAAACCGTAA
Oligo sequence:
1 ATGGACGCGCTGACCAC 17
2 CCGGGAAACGGTTCAGGAGCGCGGTGTGTTTTTTGATCGGCAGCGTGGTCAGCGCGTCCA 60
3 TCCTGAACCGTTTCCCGGAAACCCGTTTCGTTACGCAACTGGCGAAGAAACGCGCGAGCT 60
4 TCTTCAAACTGTGCCGGGGTCAGATAGTGACCGAAAACGATCCAGCTCGCGCGTTTCTTC 60
5 CCCGGCACAGTTTGAAGATATGGACTTCTTCACCAATCGCTTTAATGCCATCCTCGATAT 60
6 CGTCCATGAGCGCAACCTCATAACGACCCACTTTCCACATATCGAGGATGGCATTAAAGC 60
7 GGTTGCGCTCATGGACGGTGAACTCACCTCTGAACACGAAACCATTCTGAAGGCGCTGGA 60
8 TTGGTGAGGTCCGGAACGTCCTGGATACGTGCGTAATCGAGTTCCAGCGCCTTCAGAATG 60
9 CGTTCCGGACCTCACCAAACCGGGTCTCATCGTTCTGGACATGGATTCTACCGCGATTCA 60
10 ACACCCGCCAGCTTCGCGATTTCGTCGATGCATTCGATCTGAATCGCGGTAGAATCCATG 60
11 CGAAGCTGGCGGGTGTCGGTGAGGAAGTTGCGGAAGTTACCGAACGTGCTATGCAGGGCG 60
12 CAGTTTAGAAACACGGAGACGCAGAGACTGTTCGAAATCCAGTTCGCCCTGCATAGCACG 60
13 CGTCTCCGTGTTTCTAAACTGAAGGATGCACCGGAACAGATCCTGAGCCAAGTTCGTGAA 60
14 GTCGCAACGAGCTCTGGCAGTTCCGGCATCAGCGGCAGGGTTTCACGAACTTGGCTCAGG 60
15 CCAGAGCTCGTTGCGACCCTGCACGCATTCGGTTGGAAGGTAGCAATCGCCTCCGGTGGT 60
16 CCAGAGAGAGCTGCTCTTTCAGGTAGTCGCTGAAGTAGGTAAAACCACCGGAGGCGATTG 60
17 GAAAGAGCAGCTCTCTCTGGACTATGCGCAGTCTAACACCCTCGAAATTGTTTCTGGTAA 60
18 GCGCGGAGACAACTTCACCGAGAACCTGACCAGTGAGTTTACCAGAAACAATTTCGAGGG 60
19 GTGAAGTTGTCTCCGCGCAGACCAAAGCGGACATCCTGCTGACGCTCGCCCAGCAGTATG 60
20 TTCGCGCCATCGCCAACCGCAACGGTGTTGTGAATTTCAACGTCATACTGCTGGGCGAGC 60
21 TGGCGATGGCGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTGGGTGTGGCCTACCA 60
22 ACGAACCGCCGTTTGCGCCTTTGCTTCGACTTTCGGTTTCGCGTGGTAGGCCACACCCAG 60
23 GCAAACGGCGGTTCGTTTTGCGGGTCTGGGTGGTGTCGTTTGTATTCTGTCTGCCGCGCT 60
24 TTACGGTTTAGATTTCCAAGACAGTTTTTGCTGCGCCACCAGCGCGGCAGACAGAAT 57
*GC% Content for gene: 56.3%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Upstream:
TACTTCCAATCCATGGACGCGCTGACCA
Downstream (reverse complemented):
TATCCACCTTTACTGTTACGGTTTAGATTTCCAAGAGAG
*Note that this insert contains code that codes for a cut by BsaI.
We will be ordering from Shelby-Dax Garibay.
Resources:
See ProtocolTargetDiscoveryVDS.docx for moreEtiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
Databases of genes/organisms:
http://www.niaid.nih.gov/Pages/default.aspx
http://eupathdb.org/eupathdb/
https://patricbrc.vbi.vt.edu/portal/portal/patric/Home
http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes
http://tubic.tju.edu.cn/deg/
http://csgid.org/csgid/cake/pages/community_request_gateway
http://tdrtargets.org/
http://gsc.jcvi.org/status.shtml
Possible inhibitors:
http://www.ncbi.nlm.nih.gov/pubmed/9430431
Comparison of phosphoserine and amino acid binding in another bacteria:
http://www.rcsb.org/pdb/explore/explore.do?structureId=1L7P
Comparison of protein to other bacteria with cation in active site:
http://www.rcsb.org/pdb/explore.do?structureId=1L7M
Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)
http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf
Primer Overlap PCR Articles
HooverLubkowski_PCRoverlapcloninggnf042.pdf
StemmerPCRoverlapGene1995.pdf
Is my target good for Virtual Screening programs?
Reynolds_THermodynamicsLigandBinding_MedChemLett2011.pdf